This competitive renewal application capitalizes on two key findings of the previous funding cycle: (i) the definitive demonstration of an essential role of the epsilon isoform of PKC in mediating various forms of cardioprotection, including nitric oxide (NO) donor- and ischemia-induced preconditioning. This finding is supported by and in agreement with reports from multiple other investigators in the field; and (ii) the observation that PKCepsilon does not function in isolation during cardioprotection. Instead, it recruits the participation of other molecules to form a cardioprotective sub-proteome. This knowledge in hand, we are now focusing on the next challenge: to determine the subcellular targets of PKCepsilon that are the effectors of the cardioprotective process. Recent developments in the field of myocardial ischemia have solidified the mitochondrion as a critical regulator of cell survival. While the role of mitochondria as energy-producing organelles is well established in the heart and other organs, the emergent, and less well understood, role of mitochondria as signaling hubs is the focus of this application. In particular, prevention of mitochondrial permeability transition (MPT) -an opening of non-selective pores that leads to mitochondrial dysfunction and cell death--has been shown to be cardioprotective. Interestingly, we identified the cardiac isoform of the adenine nucleotide translocase (ANT1) as a member of the PKCepsilon sub-proteome. This was a very exciting finding because ANT1 is a central pore-forming unit of the MPT pore, and many recent investigations have implicated ANT1 in MPT. Taken with the insight gained from our previous award documenting the role of PKCepsilon as a protective molecule, the presence of ANT1 in the PKCepsilon sub-proteome suggested a mechanism whereby PKCs signaling may impinge on the mitochondria to influence the propensity for MPT during ischemic insult. Consistent with this notion, our preliminary data show that administration of the drug atractyloside, which binds to ANT1 and induces pore formation, abolishes cardioprotection. Importantly, our preliminary data confirms co-localization of ANT1 with PKCepsilon in the inner membrane of the mitochondria by co-immunoprecipitation and LC/MS/MS. Furthermore, we have demonstrated that ANT1 is a substrate for PKCepsilon in vitro. Phosphorylation of ANT1 by PKCepsilon appears to occur on the third matrix loop of ANT1 on one or both of two PKC consensus sites that flank a key regulatory motif(the RRRMMM motif) of the molecule, suggesting that PKCepsilon may directly modify a central component of the MPT pore. Substantiating this concept, our preliminary data show that inhibition of PKCs is sufficient to block cardioprotective attenuation of mitochondrial swelling and reduction in myocardial infarct size. Using a comprehensive approach that combines a well-established animal model of cardioprotection, protein chemistry, mass spectrometry, and membrane biochemistry, this proposal will conclusively examine the novel hypothesis that a critical task of PKCepsilon in NO donor-induced pharmacological preconditioning is to attenuate mitochondrial permeability transition. This action involves, in part, direct modification of ANT1, thereby reducing the poreforming ability of this molecule. Information to be gained will afford opportunities to further our understanding of the molecular basis of preconditioning and the cellular targets that are critical for the manifestation of cardioprotection.